Keigo Yoshizaki

Critical role of heparin binding domains of ameloblastin for dental epithelium cell adhesion and ameloblastoma proliferation

AMBN (ameloblastin) is an enamel matrix protein that regulates cell adhesion, proliferation, and differentiation of ameloblasts. In AMBN-deficient mice, ameloblasts are detached from the enamel matrix, continue to proliferate, and form a multiple cell layer; often, odontogenic tumors develop in the maxilla with age. However, the mechanism of AMBN functions in these biological processes remains unclear. By using recombinant AMBN proteins, we found that AMBN had heparin binding domains at the C-terminal half and that these domains were critical for AMBN binding to dental epithelial cells. Overexpression of full-length AMBN protein inhibited proliferation of human ameloblastoma AM-1 cells, but overexpression of heparin binding domain-deficient AMBN protein had no inhibitory effect. In full-length AMBN-overexpressing AM-1 cells, the expression of Msx2, which is involved in the dental epithelial progenitor phenotype, was decreased, whereas the expression of cell proliferation inhibitors p21 and p27 was increased. We also found that the expression of enamelin, a marker of differentiated ameloblasts, was induced, suggesting that AMBN promotes odontogenic tumor differentiation. Thus, our results suggest that AMBN promotes cell binding through the heparin binding sites and plays an important role in preventing odontogenic tumor development by suppressing cell proliferation and maintaining differentiation phenotype through Msx2, p21, and p27.

Neurotrophic factor neurotrophin-4 regulates ameloblastin expression via full-length TrkB

Neurotrophic factors play an important role in the development and maintenance of not only neural but also nonneural tissues. Several neurotrophic factors are expressed in dental tissues, but their role in tooth development is not clear. Here, we report that neurotrophic factor neurotrophin (NT)-4 promotes differentiation of dental epithelial cells and enhances the expression of enamel matrix genes. Dental epithelial cells from 3-day-old mice expressed NT-4 and three variants of TrkB receptors for neurotrophins (full-length TrkB-FL and truncated TrkB-T1 and -T2). Dental epithelial cell line HAT-7 expressed these genes, similar to those in dental epithelial cells. We found that NT-4 reduced HAT-7 cell proliferation and induced the expression of enamel matrix genes, such as ameloblastin (Ambn). Transfection of HAT-7 cells with the TrkB-FL expression construct enhanced the NT-4-mediated induction of Ambn expression. This enhancement was blocked by K252a, an inhibitor for Trk tyrosine kinases. Phosphorylation of ERK1/2, a downstream molecule of TrkB, was induced in HAT-7 cells upon NT-4 treatment. TrkB-FL but not TrkB-T1 transfection increased the phosphorylation level of ERK1/2 in NT-4-treated HAT-7 cells. These results suggest that NT-4 induced Ambn expression via the TrkB-MAPK pathway. The p75 inhibitor TAT-pep5 decreased NT-4-mediated induction of the expression of Ambn, TrkB-FL, and TrkB-T1, suggesting that both high affinity and low affinity neurotrophin receptors were required for NT-4 activity. We found that NT-4-null mice developed a thin enamel layer and had a decrease in Ambn expression. Our results suggest that NT-4 regulates proliferation and differentiation of the dental epithelium and promotes production of the enamel matrix.

Platelet-derived Growth Factor Receptor Regulates Salivary Gland Morphogenesis via Fibroblast Growth Factor Expression

A coordinated reciprocal interaction between epithelium and mesenchyme is involved in salivary gland morphogenesis. The submandibular glands (SMGs) of Wnt1-Cre/R26R mice have been shown positive for mesenchyme, whereas the epithelium is β-galactosidase-negative, indicating that most mesenchymal cells are derived from cranial neural crest cells. Platelet-derived growth factor (PDGF) receptor α is one of the markers of neural crest-derived cells. In this study, we analyzed the roles of PDGFs and their receptors in the morphogenesis of mouse SMGs. PDGF-A was shown to be expressed in SMG epithelium, whereas PDGF-B, PDGFRα, and PDGFRβ were expressed in mesenchyme. Exogenous PDGF-AA and -BB in SMG organ cultures demonstrated increased levels of branching and epithelial proliferation, although their receptors were found to be expressed in mesenchyme. In contrast, short interfering RNA for Pdgfa and -b as well as neutralizing antibodies for PDGF-AB and -BB showed decreased branching. PDGF-AA induced the expression of the fibroblast growth factor genes Fgf3 and -7, and PDGF-BB induced the expression of Fgf1, -3, -7, and -10, whereas short interfering RNA for Pdgfa and Pdgfb inhibited the expression of Fgf3, -7, and -10, indicating that PDGFs regulate Fgf gene expression in SMG mesenchyme. The PDGF receptor inhibitor AG-17 inhibited PDGF-induced branching, whereas exogenous FGF7 and -10 fully recovered. Together, these results indicate that fibroblast growth factors function downstream of PDGF signaling, which regulates Fgf expression in neural crest-derived mesenchymal cells and SMG branching morphogenesis. Thus, PDGF signaling is a possible mechanism involved in the interaction between epithelial and neural crest-derived mesenchyme.

Sprouty2 controls proliferation of palate mesenchymal cells via fibroblast growth factor signaling.

Cleft palate is one of the most common craniofacial deformities. The fibroblast growth factor (FGF) plays a central role in reciprocal interactions between adjacent tissues during palatal development, and the FGF signaling pathway has been shown to be inhibited by members of the Sprouty protein family. In this study, we report the incidence of cleft palate, possibly caused by failure of palatal shelf elevation, in Sprouty2-deficient (KO) mice. Sprouty2-deficient palates fused completely in palatal organ culture. However, palate mesenchymal cell proliferation estimated by Ki-67 staining was increased in Sprouty2 KO mice compared with WT mice. Sprouty2-null palates expressed higher levels of FGF target genes, such as Msx1, Etv5, and Ptx1 than WT controls. Furthermore, proliferation and the extracellular signal-regulated kinase (Erk) activation in response to FGF was enhanced in palate mesenchymal cells transfected with Sprouty2 small interfering RNA. These results suggest that Sprouty2 regulates palate mesenchymal cell proliferation via FGF signaling and is involved in palatal shelf elevation.

Teneurin-4 is a Novel Regulator of Oligodendrocyte Differentiation and Myelination of Small Diameter Axons in the CNS

Myelination is essential for proper functioning of the CNS. In this study, we have identified a mouse mutation, designated furue, which causes tremors and hypomyelination in the CNS, particularly in the spinal cord, but not in the sciatic nerve of the PNS. In the spinal cord of the furue mice, myelination of small-diameter axons was dramatically reduced, and differentiation of oligodendrocytes, the myelin-forming cells in the CNS, was inhibited. We subsequently found that the furue mutation was associated with a transgene insertion into the teneurin-4 (Ten-4, Ten-m4/Odz4) gene, encoding a transmembrane protein of unknown function. Ten-4 was strongly expressed in the spinal cord of wild-type mice and was induced during normal oligodendrocyte differentiation. In contrast, in the furue mice, the expression of Ten-4 was absent. Differentiation and cellular process formation of oligodendrocytes were inhibited in primary cell culture from the furue mice. Cell differentiation and process formation were also inhibited in the oligodendrocyte progenitor cell line CG-4 after suppression of Ten-4 expression by shRNA. Furthermore, Ten-4 positively regulated focal adhesion kinase, an essential signaling molecule for oligodendrocyte process formation and myelination of small-diameter axons. These findings suggest that Ten-4 is a novel regulator of oligodendrocyte differentiation and that it plays a critical role in the myelination of small-diameter axons in the CNS.

TGF-β3 induces ectopic mineralization in fetal mouse dental pulp during tooth germ development

Several members of the transforming growth factor (TGF)‐β superfamily are expressed in developing teeth from the initiation stage through adulthood. Of those, TGF‐β1 regulates odontoblast differentiation and dentin extracellular matrix synthesis. However, the molecular mechanism of TGF‐β3 in dental pulp cells is not clearly understood. In the present study, beads soaked with human recombinant TGF‐β3 induced ectopic mineralization in dental pulp from fetal mouse tooth germ samples, which increased in a dose‐dependent manner. Further, TGF‐β3 promoted mRNA expression, and increased protein levels of osteocalcin (OCN) and type I collagen (COL I) in dental pulp cells. We also observed that the expression of dentin sialophosphoprotein and dentin matrix protein 1 was induced by TGF‐β3 in primary cultured dental pulp cells, however, not in calvaria osteoblasts, whereas OCN, osteopontin and osteonectin expression was increased after treatment with TGF‐β3 in both dental pulp cells and calvaria osteoblasts. Dentin sialoprotein was also partially detected in the vicinity of TGF‐β3 soaked beads in vivo. These results indicate for the first time that TGF‐β3 induces ectopic mineralization through upregulation of OCN and COL I expression in dental pulp cells, and may regulate the differentiation of dental pulp stem cells to odontoblasts.

Gene evolution and functions of extracellular matrix proteins in teeth

Abstract The extracellular matrix (ECM) not only provides physical support for tissues, but it is also critical for tissue development, homeostasis and disease. Over 300 ECM molecules have been defined as comprising the “core matrisome” in mammals through the analysis of whole genome sequences. During tooth development, the structure and functions of the ECM dynamically change. In the early stages, basement membranes (BMs) separate two cell layers of the dental epithelium and the mesenchyme. Later in the differentiation stages, the BM layer is replaced with the enamel matrix and the dentin matrix, which are secreted by ameloblasts and odontoblasts, respectively. The enamel matrix genes and the dentin matrix genes are each clustered in two closed regions located on human chromosome 4 (mouse chromosome 5), except for the gene coded for amelogenin, the major enamel matrix protein, which is located on the sex chromosomes. These genes for enamel and dentin matrix proteins are derived from a common ancestral gene, but as a result of evolution, they diverged in terms of their specific functions. These matrix proteins play important roles in cell adhesion, polarity, and differentiation and mineralization of enamel and dentin matrices. Mutations of these genes cause diseases such as odontogenesis imperfecta (OI) and amelogenesis imperfecta (AI). In this review, we discuss the recently defined terms matrisome and matrixome for ECMs, as well as focus on genes and functions of enamel and dentin matrix proteins.

Connexin43 plays an important role in lung development.

OBJECTIVES Connexin43 (Cx43) is one of the proteins associated with gap junction. Connexin43 knockout mice die after birth owing to hypoplastic lungs. The purpose of this study was to analyze the hypoplastic lung of Cx43 knockout mice to clarify the role of the Cx43 during lung development. METHODS Adult hetero Cx43 mice were mated. Newborn mice were divided into the following groups: wild, hetero, and knockout. Total RNA was extracted from the right lung, and the left lung was fixed for immunohistochemical staining. The mRNA expression of surfactant protein C, aquaporin-5, and alpha-smooth muscle actin were analyzed by reverse transcriptase polymerase chain reaction. H&E and immunohistochemical staining for those markers were performed. RESULTS The mRNA expression of aquaporin-5, surfactant protein C, and alpha-smooth muscle actin was significantly lower in knockout mice than that in the wild and hetero mice. H&E staining in the knockout mice showed narrow airspaces and thicker interalveolar septae. Immunohistochemical staining in all markers showed the formation of alveoli to be delayed in the knockout mice. CONCLUSION Based on these findings, Cx43 is closely related to alveolar and vascular formation during lung development.

Retrospective Evaluation of Treatment Outcome in Japanese Children With Complete Unilateral Cleft Lip and Palate. Part 1: Five-Year-Olds' Index for Dental Arch Relationships

Objective: To evaluate the dental arch relationships of Japanese children with complete unilateral cleft lip and palate (UCLP) and to examine the 5-year-olds’ index for its validity. Design: Retrospective study and comparison with previous reports. Subjects: One hundred thirty-six children with complete UCLP who received primary cheiloplasty and palatoplasty in the Kyushu University Hospital from 1966 to 1999. Materials: Dental models taken from children 53 to 67 months of age and their cephalograms. Methods: Study models were assessed using five scores; 1 = excellent, 2 = good, 3 = fair, 4 = poor, and 5 = very poor, in accordance with the 5-year-olds’ index and also evaluated using Huddart and Bodenhams numerical classification. Dental arch widths, three-dimensional maxillary dental arch form, and lateral cephalograms were traced and measured. The outcome by 5-year-olds’ index was compared with Huddart and Bodenhams numerical classification, dental arch dimensions, and cephalometric measurements. Results: Occlusal outcome evaluated by the 5-year-olds’ index was rated 2.95, which was classified as fair. This index rating showed a significant relationship with numerical classification and dental arch length, but not with dental arch width. The index showed a relationship with mandibular form and position, but not with maxillary position. Conclusion: The occlusal outcome of the cases with UCLP was fair as evaluated using the 5-year-olds’ index. The index evaluates the anteroposterior relationship of maxillary/mandibular dental arches but does not evaluate the collapse of maxillary segments.

Interaction between fibronectin and β1 integrin is essential for tooth development.

The dental epithelium and extracellular matrix interact to ensure that cell growth and differentiation lead to the formation of teeth of appropriate size and quality. To determine the role of fibronectin in differentiation of the dental epithelium and tooth formation, we analyzed its expression in developing incisors. Fibronectin mRNA was expressed during the presecretory stage in developing dental epithelium, decreased in the secretory and early maturation stages, and then reappeared during the late maturation stage. The binding of dental epithelial cells derived from postnatal day-1 molars to a fibronectin-coated dish was inhibited by the RGD but not RAD peptide, and by a β1 integrin-neutralizing antibody, suggesting that fibronectin-β1 integrin interactions contribute to dental epithelial-cell binding. Because fibronectin and β1 integrin are highly expressed in the dental mesenchyme, it is difficult to determine precisely how their interactions influence dental epithelial differentiation in vivo. Therefore, we analyzed β1 integrin conditional knockout mice (Intβ1lox-/lox-/K14-Cre) and found that they exhibited partial enamel hypoplasia, and delayed eruption of molars and differentiation of ameloblasts, but not of odontoblasts. Furthermore, a cyst-like structure was observed during late ameloblast maturation. Dental epithelial cells from knockout mice did not bind to fibronectin, and induction of ameloblastin expression in these cells by neurotrophic factor-4 was inhibited by treatment with RGD peptide or a fibronectin siRNA, suggesting that the epithelial interaction between fibronectin and β1 integrin is important for ameloblast differentiation and enamel formation.